Human wild‐type and D76N β2‐microglobulin variants are significant proteotoxic and metabolic stressors for transgenic C. elegans

Abstract β2‐microglobulin (β2‐m) is a plasma protein derived from physiological shedding of the class I major histocompatibility complex (MHCI), causing human systemic amyloidosis either due to persistently high concentrations of the wild‐type (WT) protein in hemodialyzed patients, or in presence of mutations, such as D76N β2‐m, which favor protein deposition in the adulthood, despite normal plasma levels. Here we describe a new transgenic Caenorhabditis elegans (C. elegans) strain expressing human WT β2‐m at high concentrations, mimicking the condition that underlies dialysis‐related amyloidosis (DRA) and we compare it to a previously established strain expressing the highly amyloidogenic D76N β2‐m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with β2‐m levels rather than with the presence of mutations, being more pronounced in WT β2‐m worms. β2‐m expression also has a deep impact on the nematodes' proteomic and metabolic profiles. Most significantly affected processes include protein degradation and stress response, amino acids metabolism, and bioenergetics. Molecular alterations are more pronounced in worms expressing WT β2‐m at high concentration compared to D76N β2‐m worms. Altogether, these data show that β2‐m is a proteotoxic protein in vivo also in its wild‐type form, and that concentration plays a key role in modulating pathogenicity. Our transgenic nematodes recapitulate the distinctive features subtending DRA compared to hereditary β2‐m amyloidosis (high levels of non‐mutated β2‐m vs. normal levels of variant β2‐m) and provide important clues on the molecular bases of these human diseases.

1D 1H NMR spectra was acquired with water peak suppression and a standard NOESY (1) pulse sequence (Bruker pulse program noesypr1d) using 32 scans, 65536 data points, a spectral width of 13888.9Hz, an acquisition time of 2.7 s, a relaxation delay of 10 s and a mixing time of 0.1 s.
The 1D NMR spectra were processed and analyzed with the software ACD-Labs Spectrus Processor 2020.2.0.After FID apodization with an exponential multiplication window function (LB 0.3), all spectra were phased, Fourier-transformed and chemical-shift referenced.
Gradient enhanced magnitude COSY pulse sequence (cosygpprqf supplied by Bruker) with a presaturation during relaxation delay was used.Spectra were collected with 2048 points in t2 and 256 points in t1 over a sweep width of 9615.385Hz, with 8 scans per t1 value.The acquisition times were fixed to 0.11 s in F2 and 0.027s in F1, the relaxation delay to 2 s.The TOCSY pulse sequence (mlevphpr.2supplied by Bruker) included: coherence transfer during a multiple-pulse spin-lock period, solvent suppression by presaturation, applied along the 2s relaxation delay, and mixing time of 80 ms.Spectra were collected with 2048 points in t2 and 256 points in t1 over a sweep width of 11363.637Hz, with 8 scans per t1 value.The acquisition times were fixed to 0.09 s in F2 and 0. 0.01s in F1.The resulting COSY and TOCSY spectra were processed in Topspin 4.1.0using standard methods, with sine-squared apodization in both dimensions and zero filling in t1 to yield a transformed 2D dataset of 1024 by 1024 points.The 1H, 13C-HSQC pulse sequence (hsqcetgpsisp2.2supplied by Bruker) using sensitivity improvement and shaped-pulses for all 180-degree pulses on 1Hchannel, with decoupling during acquisition.Spectra were collected with 2048 points in t2 and 128 points in t1 over a sweep width of 9615.385Hz for 1H and 31691.145Hz for 13C, with 16 scans per t1 value.
The acquisition times were fixed to 0.11s in F2 and 0.002s in F1, the relaxation delay to 2 s.
The resulting HSQC spectra were processed in Topspin 4.1.0using standard methods, with sine-squared apodization in both dimensions, 0th order phase correction in 1H of 180 degrees and zero filling in t1 to yield a transformed 2D dataset of 1024 by 1024 points.
Table S1.Differentially represented proteins in D76N vs ctrl and WT vs ctrl comparisons.= Significantly altered proteins emerging from both comparisons with the same direction of deregulation; ≠ significantly altered proteins emerging from both comparisons with the same deregulation; CY, cytoplasm; N, nucleus; M, mitochondrion; CS, cytoskeleton; R, ribosome; ER, endoplasmic reticulum; Me, cell membrane; Secr., Secreted.Proteins without a Gene Ontology or Uniprot annotations for cellular location are shown as 'Unclassified'.Table S3.Results of the joint pathway analysis of proteomics and metabolomics data.

WT vs ctrl
Pathway Hits Match status -log10(p) Impact

Figure S1 .
Figure S1.Relative quantification by qPCR of D76N and WT β2-m DNA extracted from the two C. elegans strains.Genomic DNA was extracted from 5'000 nematode's samples of both WT β2-m and D76N β2-m strains after lysis in 5 volumes of worm genomic DNA lysis buffer (10mM Tris pH 7.5, 2mM EDTA, 0.5% SDS) and proteinase K 0,1 mg/ml.Organic extraction was performed with phenol/chloroform/isoamyl alcohol and qPCR on DNA samples extracted from both strains was performed using a Quantifast SYBR Green PCR (QIAGEN, 204054) together with β2-m and cdc42 (Cell Division Cycle) primers.Relative quantification was performed using Cdc-42 as housekeeping gene.Data shown in the graph correspond to normalized Ct values of six replicates from two independent experiments.β2-m gene quantification of wildtype and mutated D76N protein showed no significant differences between the two strains according to the Mann-Whitney test (GraphPad Prism).

Figure S2 .
Figure S2.A) and B) Western blots for detection of β2-m and actin.Delimited portions (dotted squares) are used in Figure 1C and in Figure 2A respectively.(M= Molecular weight standard: Precision Plus Western C, BioRad).C) Western blots for detection of β2-m.Delimited portion (dotted squares) is used in Figure 2C (M= Molecular weight standard: Precision Plus Western C, BioRad; SM= starting material loaded into gel filtration column).Please note that the upper parts of membranes developed with anti-β2-m in B and C were covered during the chemiluminescence signal capture to enhance the specific β2m signal versus the nonspecific bands due to the secondary antibody as previously shown (1).

Table S2 . MS-identified and quantified metabolites.
Results of the unpaired non-parametric Mann-Withney test applied to each class of quantified metabolites.= Same direction of change; ≠ different direction of change; + Significantly altered in one comparison; ++ Significantly altered in both comparison WT vs ctrl (over-represented proteins, n = 300)